This protocol describes the chemical dimerization system to induce protein condensates on telomeres. It can be easily adapted to induce condensates on other genomic loci, and is suitable for probing the formation and function of chromatin associated condensates. This method offers temporal resolution required for live cell imaging and maintains phase separation in the population of cells for biochemical assays.
To begin, dissolve dimerizers in DMSO at 10 millimolar and store them in plastic microcentrifuge tubes at minus 80 degrees Celsius for long-term storage. Dilute an aliquot of 10 millimolar dimerizer in imaging medium to a stock concentration of 10 micromolar and store it at minus 20 degrees Celsius. When ready to use, dilute dimerizers to a final working concentration of 100 nanomolar in growth medium or imaging medium.
Seed 10 to the fifth cells in 12 millimeter diameter circular cover glasses coated with Poly-D-Lysine in a six-well plate. Then transfect the cells with the Halo-GFP-TRF1 and mCherry-eDHFR-SIM or mCherry-eDFR-SIM mutant plasmids and wait for 24 to 48 hours before proceeding to immunofluorescence. Add the diluted 100 nanomolar dimerizers to the cells and incubate them in 37 degrees Celsius for four to five hours.
After the incubation, fix the cells in PBS solution containing 4%formaldehyde and 0.1%Triton X-100 for 10 minutes at room temperature to permeabilize the cells. Then wash the cells three times with PBS. Wash cover slips twice with 50 microliters of TBS-Tx and once with 50 microliters of antibody dilution buffer.
Incubate each cover slip with 50 microliters of primary anti-PML, anti-SUMO-1, or anti-SUMO-2 and 3 antibody at four degrees Celsius in a humidified chamber overnight. Wash cover slips three times with antibody dilution buffer to remove unbound primary antibody, then incubate the cells with secondary antibody for one hour in a dark box at room temperature. After staining, wash cover slips three times with TBS-Tx.
Label slides by adding two microliters of one microgram per milliliter DAPI, then flip the cover slips over and place them onto the DAPI drop. Aspirate extra fluid from the edge of the cover slip. Seal the slide with nail polish, allow it to dry, and rinse the top of the cover slip with water.
Place the slides in the freezer until imaging. Seed 10 to the fifth cells on 12 millimeter circular cover glasses coated with Poly-D-Lysine in a six-well plate and transfect them with Halo-TRF1 and mCherry-eDHFR-SIM or mCherry-eDHFR-SIM mutant plasmids. Fix the cells with 4%formaldehyde for 10 minutes at room temperature and wash them four times with PBS.
For IF-FISH, stain the cells with primary and secondary antibodies and refix them with 4%formaldehyde for 10 minutes at room temperature, then wash them four times with PBS and dehydrate the cover slips in an ethanol series. Incubate the cover slips with the 488 telomere C-PNA probe in five microliters of hybridization solution at 75 degrees Celsius for five minutes, then incubate the cover slips overnight in a humidified chamber at room temperature. Wash the cover slips three times with wash buffer for two minutes per wash at room temperature and mount them with one microgram per milliliter DAPI in mounting media for imaging.
For live imaging, mount the cover slips in magnetic chambers on a heated stage in an environmental chamber, maintaining the cells in one milliliter of imaging medium without phenol red. Set up the microscope and environmental control apparatus as described in the text manuscript. Locate cells with a bright GFP signal on the telomeres and diffusive mCherry signal in the cytosol.
Find around 20 cells, memorizing each position with the XYZ information, and set up parameters for time-lapse imaging. Use 0.5 micrometer spacing for a total of eight micrometers in Z and five minute time interval for two to four hours for both GFP and mCherry channels. Use 30%of 594 nanometer and 50%of 488 nanometer power intensity, with exposure times of 200 milliseconds and a camera gain of 300.
Start imaging and take one time loop as pre-dimerization. Then pause imaging, add 0.5 milliliters of imaging media containing 15 microliters of 10 micromolar dimerizer to the imaging chamber, taking care not to touch the stage, and resume imaging. When ready to reverse dimerization, pause imaging and add 0.5 milliliters of imaging media containing two microliters of 100 millimolar stock TMP to the imaging chamber.
Continue imaging the cells for one to two hours. For fixed imaging, use the same microscope setup as live imaging, but without the stage heating. Locate around 30 to 50 cells with the red signal to select for transfected cells.
Acquire images with 0.3 micrometer spacing for a total of eight micrometers in Z.Use 80%of 647 nanometer, 80%of 561 nanometer, and 70%of 488 nanometer power intensity, with exposure times of 600 milliseconds and a camera gain of 300. Telomeric localization of SUMO was identified using telomere DNA FISH and SUMO protein immunofluorescence. Cells with SIM recruitment enriched SUMO-1 and SUMO-2 and 3 compared to cells with SIM mutant recruitment, indicating that SIM dimerization induced SUMO enrichment on telomeres depends on SUMO-SIM interactions.
A time-lapse movie of TRF-1 and SIM after dimerization is shown here. SIM was successfully recruited to telomeres, and both SIM and TRF-1 foci became larger and brighter, as predicted for liquid droplet formation and growth. In addition, fusion of TRF-1 foci was observed, which led to telomere clustering, as shown in the reduced telomere number and increased telomere intensity over time.
In contrast, SIM mutant was recruited to telomeres after dimerization, but did not induce any droplet formation or telomere clustering, indicating that phase separation and telomere clustering is driven by SUMO-SIM interactions. The reversal of phase separation and telomere clustering was observed after adding excess free TMP. Telomere number increased and telomere intensity decreased over time.
Representative images of APBs identified by telomere DNA FISH and PML protein IF are shown here. Cells with SIM recruited have more APBs than cells with SIM mutant recruited, suggesting dimerization induced condensates are indeed APBs. When performing this protocol, do not expose light sensitive dimerizers to light.
Work in a dark room with a lamp that emits red light and wrap cells with aluminum foil during incubation. Following this procedure, proximity labeling can be used to generate a comprehensive list of components in the induced condensate. Live imaging can be used to follow the dynamics of components in the condensate.
These methods can help determine the roles of governing condensate composition control.
